New thioredoxins and glutaredoxins as electron donors of 3'-phosphoadenylylsulfate reductase.
(1/419)
Reduction of inorganic sulfate to sulfite in prototrophic bacteria occurs with 3'-phosphoadenylylsulfate (PAPS) as substrate for PAPS reductase and is the first step leading to reduced sulfur for cellular biosynthetic reactions. The relative efficiency as reductants of homogeneous highly active PAPS reductase of the newly identified second thioredoxin (Trx2) and glutaredoxins (Grx1, Grx2, Grx3, and a mutant Grx1C14S) was compared with the well known thioredoxin (Trx1) from Escherichia coli. Trx1, Trx2, and Grx1 supported virtually identical rates of sulfite formation with a Vmax ranging from 6.6 units mg-1 (Trx1) to 5.1 units mg-1 (Grx1), whereas Grx1C14S was only marginally active, and Grx2 and Grx3 had no activity. The structural difference between active reductants had no effect upon Km PAPS (22.5 microM). Grx1 effectively replaced Trx1 with essentially identical Km-values: Km trx1 (13.7 microM), Km grx1 (14.9 microM), whereas the Km trx2 was considerably higher (34.2 microM). The results agree with previous in vivo data suggesting that Trx1 or Grx1 is essential for sulfate reduction but not for ribonucleotide reduction in E. coli. (+info)
Direct NMR observation of the Cys-14 thiol proton of reduced Escherichia coli glutaredoxin-3 supports the presence of an active site thiol-thiolate hydrogen bond.
(2/419)
The active site of Escherichia coli glutaredoxin-3 (Grx3) consists of two redox active cysteine residues in the sequence -C11-P-Y-C14-H-. The 1H NMR resonance of the cysteine thiol proton of Cys-14 in reduced Grx3 is observed at 7.6 ppm. The large downfield shift and NOEs observed with this thiol proton resonance suggest the presence of a hydrogen bond with the Cys-11 thiolate, which is shown to have an abnormally low pKa value. A hydrogen bond would also agree with activity data of Grx3 active site mutants. Furthermore, the activity is reduced in a Grx3 H15V mutant, indicating electrostatic contributions to the stabilization of the Cys-11 thiolate. (+info)
17beta-estradiol induces protein thiol/disulfide oxidoreductases and protects cultured bovine aortic endothelial cells from oxidative stress.
(3/419)
OBJECTIVE: To examine whether or not estrogens induced the expression of protein thiol/disulfide oxidoreductases such as protein disulfide isomerase (PDI), thioredoxin (Trx), Trx reductase, and glutaredoxin (Grx) in vascular endothelial cells. METHODS: The regenerative effects of the protein thiol/disulfide oxidoreductases, PDI, Trx and Grx, on oxidatively damaged proteins were assayed using H2O2-inactivated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reporter enzyme. The induction of protein thiol/disulfide oxidoreductases and the accumulation of protein adducts generated by lipid peroxidation were examined by Western blotting in estrogen-treated bovine aortic endothelial cells (BAECs). RESULTS: Reduced PDI, Trx and Grx regenerated the H2O2-inactivated GAPDH in vitro. The levels of these protein disulfide oxidoreductases in BAECs were increased by pretreatment with 0.01-10 micromol/l 17beta-estradiol, the largest increase (about fourfold of the control) being found for PDI. Other sex hormones such as progesterone and testosterone did not affect the contents of these oxidoreductases in BAECs. 4-Hydroxy-2-nonenal (HNE)-protein adducts, which are generated by lipid peroxidation, were accumulated in BAECs exposed to paraquat, whereas the pretreatment of BAECs with 17beta-estradiol suppressed their accumulation. CONCLUSIONS: The estrogen-mediated induction of the protein thiol/disulfide oxidoreductases such as PDI, Trx, Trx reductase and Grx suggested a possible involvement of these oxidoreductases in the antioxidant protection of estrogen observed in the vascular system. (+info)
Induction of thioredoxin, thioredoxin reductase and glutaredoxin activity in mouse skin by TPA, a calcium ionophore and other tumor promoters.
(4/419)
We have measured the levels of thioredoxin, thioredoxin reductase and glutaredoxin enzyme activity in mouse skin following topical application of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator and tumor promoter. The specific activity of thioredoxin and thioredoxin reductase in extracts from normal epidermis increased by 40 and 50%, respectively, after single or multiple application of TPA. Multiple applications (twice per week for 2 weeks) of TPA increased glutaredoxin activity by >300%. Induction of the proteins lasted several days. Other PKC activators, like 12-O-retinoylphorbol 13-acetate, mezerein, 1-oleoyl-2-acetylglycerol and the calcium ionophore A23187, also induced all the enzyme activities. Phorbol and 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate, weak activators of PKC, selectively induced the thioredoxin system only and did not influence glutaredoxin activity. Multiple applications of TPA to tumor initiated (7,12-dimethyl[a]benzanthracene-treated) skin resulted in elevated levels of both the thioredoxin and glutaredoxin systems when examined 6 days after the last phorbol ester treatment. Induction of thioredoxin, thioredoxin reductase and glutaredoxin activities by TPA and calcium ionophores may play a general role in the epigenetic mechanism of tumor promotion via thiol redox control mechanisms. (+info)
Thioredoxin deficiency causes the constitutive activation of Yap1, an AP-1-like transcription factor in Saccharomyces cerevisiae.
(5/419)
Yap1 is a transcription factor that responds to oxidative stress in Saccharomyces cerevisiae. The activity of Yap1 is regulated at the level of its intracellular localization, and a cysteine-rich domain at the C terminus of Yap1 is involved in this regulation. We investigated the effects of redox-regulatory proteins, thioredoxin and glutaredoxin, on the regulation of Yap1, using the deficient mutants of these thiol-disulfide oxidoreductases. In the thioredoxin-deficient mutant (trx1Delta/trx2Delta), Yap1 was constitutively concentrated in the nucleus and the level of expression of the Yap1 target genes was high under normal conditions, while this was not the case for the glutaredoxin-deficient mutant (grx1Delta/grx2Delta). No distinct difference was observed in the levels of Yap1 protein between the wild type and trx1Delta/trx2Delta. The constitutive activation of Yap1 in trxDelta/trx2Delta was observed under aerobic conditions but not under anaerobic conditions. These findings suggest that thioredoxin has negative effects on this regulation via the redox states. We also show the synthetic lethality between yap1Delta and trx1Delta/trx2Delta mutation, but the yap1Delta/grx1Delta/grx2Delta triple mutant was viable, suggesting a difference of the functions between thioredoxin and glutaredoxin and a genetic interaction between Yap1 and thioredoxin in vivo. (+info)
Immunolocalization of glutaredoxin in the human corpus luteum.
(6/419)
Glutaredoxin (Grx) is a small protein with oxidoreductase activity which is involved in the cellular defence against oxidative stress. Corpus luteum (CL) regression has been related to the generation of reactive oxygen species (ROS). We have studied the presence of glutaredoxin in the human ovary during the ovulatory cycle using polyclonal antibodies developed against recombinant human Grx. Immunostaining was only detected between days 15 and 23 of the cycle and was localized exclusively in the corpus luteum. Grx-positive cells corresponded to granulosa-derived luteal cells (GLC) whereas the remaining luteal cell types were not immunostained. In general, Grx immunoreactivity was parallel to the functional activity of the CL. Most GLC were immunostained on days 15-16 of the cycle, whereas on days 17-19 immunoreaction was found mainly at the inner and outer aspects of the granulosa lutein layer (GLL). After this stage only isolated GLC showed Grx immunoreactivity and no reaction was found from day 23 of the cycle onward. In two CL of pregnancy that were also studied, isolated GLC showed Grx immunoreactivity. Loss of Grx immunoreactivity was coincident with the appearance of morphological signs of structural luteolysis, such as shrinkage of the GLL and the presence of apoptotic cells. These data suggest that Grx, as a cellular antioxidant, plays an important role in the mechanisms of human CL development. (+info)
Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae.
(7/419)
Glutaredoxins are members of a superfamily of thiol disulfide oxidoreductases involved in maintaining the redox state of target proteins. In Saccharomyces cerevisiae, two glutaredoxins (Grx1 and Grx2) containing a cysteine pair at the active site had been characterized as protecting yeast cells against oxidative damage. In this work, another subfamily of yeast glutaredoxins (Grx3, Grx4, and Grx5) that differs from the first in containing a single cysteine residue at the putative active site is described. This trait is also characteristic for a number of glutaredoxins from bacteria to humans, with which the Grx3/4/5 group has extensive homology over two regions. Mutants lacking Grx5 are partially deficient in growth in rich and minimal media and also highly sensitive to oxidative damage caused by menadione and hydrogen peroxide. A significant increase in total protein carbonyl content is constitutively observed in grx5 cells, and a number of specific proteins, including transketolase, appear to be highly oxidized in this mutant. The synthetic lethality of the grx5 and grx2 mutations on one hand and of grx5 with the grx3 grx4 combination on the other points to a complex functional relationship among yeast glutaredoxins, with Grx5 playing a specially important role in protection against oxidative stress both during ordinary growth conditions and after externally induced damage. Grx5-deficient mutants are also sensitive to osmotic stress, which indicates a relationship between the two types of stress in yeast cells. (+info)
Reactivity of glutaredoxins 1, 2, and 3 from Escherichia coli shows that glutaredoxin 2 is the primary hydrogen donor to ArsC-catalyzed arsenate reduction.
(8/419)
In Escherichia coli ArsC catalyzes the reduction of arsenate to arsenite using GSH with glutaredoxin as electron donors. E. coli has three glutaredoxins: 1, 2, and 3, each with a classical -Cys-Pro-Tyr-Cys- active site. Glutaredoxin 2 is the major glutathione disulfide oxidoreductase in E. coli, but its function remains unknown. In this report glutaredoxin 2 is shown to be the most effective hydrogen donor for the reduction of arsenate by ArsC. Analysis of single or double cysteine-to-serine substitutions in the active site of the three glutaredoxins indicated that only the N-terminal cysteine residue is essential for activity. This suggests that, during the catalytic cycle, ArsC forms a mixed disulfide with GSH before being reduced by glutaredoxin to regenerate the active ArsC reductase. (+info)